The opportunistic apicomplexan parasite Cryptosporidium (Crypto) causes diarrheal disease worldwide which can be devastating in AIDS patients. However, currently, there is no effective, specific therapy for AIDS- associated cryptosporidiosis. The long term goal of this project is to develop therapeutics for cryptosporidiosis in AIDS patients by targeting enzymes that synthesize O-glycans that are integral to the ability of the parasite to attach to and invade intestinal epithelial cells. Our previous and preliminary studies strongly suggest that O- glycans play a significant role in mediating infection and inducing immune responses and imply that the enzymes (UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyl transferases or ppGalNAc-Ts) which synthesize them may serve as targets for intervention for cryptosporidiosis in the immunocompromised, particularly AIDS patients. However, very little, if anything is known about these enzymes in Crypto. Our central hypothesis is that ppGalNAc-Ts that catalyze the synthesis of O-glycans on key mucin-type glycoproteins are essential for Crypto infection. The specific aim is to elucidate the functional role of ppGalNAc-Ts in Crypto infection using inhibitor identified by high throughput screening (HTS) of small molecule libraries in collaboration with National Small Molecule Screening and Medicinal Chemistry (NSRB) Core at the New the England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE-BEID). The most potent inhibitors of enzyme activity will be screened for their ability to specifically block synthesis of Crypto O-glycans. These inhibitors will then be tested for their ability to inhibit Crypto infection in vitro. At the completion of this project we expect to have expressed enzymatically active, recombinant forms of all four Cp GalNAc Ts and to have identified small molecule inhibitors which block enzyme activity, O-glycan synthesis and infection in vitro. These studies will establish the role of O-glycans and the enzymes that catalyze their synthesis in Crypto-host cell interactions and significantly advance the field. Results from this project will inform future studies on Crypto ppGalNAc-Ts and O glycans on specific glycoproteins that are critical for mediating attachment to and invasion of host cells by the parasite. Small molecule inhibitors which block Crypto ppGalNAc T enzyme activity, O-glycan synthesis and infection in vitro can be developed as potential therapeutics for AIDS-associated cryptosporidiosis in future studies.